High level expression of proteins, such as for use as biopharmaceuticals, requires proper protein folding for optimal biological activity. Overexpressed proteins are often misfolded and form biologically inactive insoluble protein aggregates, known as inclusion bodies. The formation of inclusion bodies is most often observed in the widely used E. coli expression system. In part this is due to inadequacy of prokaryotic cell protein expression machinery, which is unable to provide modifications required for biological activity of many eucaryotic proteins. For example, prokaryotic cells do not have endoplasmic reticulum, with its specific oxidative environment, as well as characteristic set of factors assisting protein folding, as well as protein modification enzymes and protein folding quality control mechanisms. However, inclusion body formation is often a problem in any eucaryotic expression system, if the level of a recombinant protein expression is practically as high as in the E. coli, for example in yeast, baculovirus or alpha virus expression systems. The main reason for that is that even though a eucaryotic cell may have an entire set of factors required for a particular protein folding and modification, the concentration of some of these factors is not sufficient to assist the modification and folding of a bulk of recombinant protein produced to an unnaturally high level. Providing factors responsible for protein folding at levels comparable with an amount of an overexpressed protein is required to solve this problem. Such factors are known as molecular chaperones.
We will be using the term molecular chaperone for any factor as far as it can assist protein folding. Ideally, a set of molecular chaperones should be provided, as chaperones act in ensembles. Therefore, a vector system allowing expression of several molecular chaperones at the same time is required for optimal protein folding assistance. In prokaryotes this task is simplified by the fact that several proteins can be expressed from the same promoter comprising an operon. In eucaryotic expression system the task is more complex, as every polynucleotide sequence encoding a protein should be provided with its own promoter or an IRES element (Wilson J. E. et al., Mol Cell Biol., 20:4990–4999, 2000).
Furthermore, in order to achieve maximum expression of a protein of interest, it is desirable to separate polynucleotide sequences encoding a protein of interest from polynucleotide sequences encoding molecular chaperones. Otherwise situated nearby in the genome promoters compete for a local pool of transcription factors. Since the employed promoters are very strong, they can deplete a local pool of transcription factors and ribonucleotide triphosphates, and a transcriptional activity from the nearby promoters, for example one controlling expression of a protein of interest and another controlling expression of a molecular chaperone could be compromised. This is particularly undesirable in respect to a promoter controlling expression of a protein of interest, since majority of the molecular chaperones are expressed to a very high level anyway. Moreover, less production of a molecular chaperone than of a protein of interest is typically required, since chaperones are recyclable and interact with a newly synthesized unfolded pool of a protein of interest, rather than with the entire pool of the protein. Therefore, placing transcriptional units encoding molecular chaperones in vicinity of each other is usually acceptable. However, separating a transcriptional unit encoding a protein of interest from any other strong transcriptional unit is preferred.
Another factor influencing performance of a multi-protein expression system is an ability to provide all the interacting proteins using as few genomes as possible. Preferably, all the polynucleotide sequences encoding for proteins interacting with each other should be provided in a single genome. For example, polynucleotide sequences encoding a protein of interest and key molecular chaperones facilitating its folding should be resident in the same plasmid DNA or in the same recombinant virus DNA, or incorporated into the same host cell genome. Performance of a multi-protein expression system is decreasing with increasing the number of genomes expressing interacting proteins due to several factors.
First, replicating genomes compete for the cell resources and expression of a protein of interest could be reduced to only about ⅓ if 3 genomes, for example 3 recombinant viruses are used at equal multiplicity of infection (m.o.i) to co-infect appropriate host cells, for example to provide for expression of a protein of interest and two molecular chaperones.
Second, due to random Poisson distribution of each type of virus between the cells, different individual cells can receive viruses of each type at different ratios. For example, it is known that for optimal folding of a protein of interest a cell should receive equal amount of each of 3 types of viruses (ratio 1:1:1), one expressing a protein of interest and another two expressing molecular chaperones. To this end, appropriate host cells can be co-infected at equal m.o.i., for example m.o.i. 10 with each type of virus. However, only a minority of cells in the population will receive all 3 viruses at the desired ratio 1:1:1. Some of the cells will receive less virus expressing a protein of interest and more of the viruses expressing molecular chaperones, in these cells expression of protein of interest would be further compromised. In other cells, which did not receive sufficient amount of at least one virus expressing a molecular chaperone, folding of protein of interest could be compromised.
Third, even if some cells received all 3 viruses at a desired ratio, for example 1:1:1, at any time point only a minority of these cells will harbor 3 types of genomes at the same ratio 1:1:1. This is due to the fact that different virus particles are not penetrating the cell simultaneously, and some are starting to replicate before the others. Typically, the process of virus adsorbtion takes about 1 h, and replication of a virus genome can take as little as a minute. Therefore, a number of the genomes of the virus, which infected cell late in adsorbtion process can be dramatically reduced compared to the number of the virus genomes, which entered the cell earlier in the process.
Similarly, different cells can be receiving different number of plasmid genomes and at different times at co-transfections. The co-transfection process differs as it involves a factor of antibiotic selective pressure to eliminate cells, which did not receive a plasmid. However, even under the selection pressure, different cells can have different copy number of the plasmids and can widely vary in the level of correctly processed protein (Keith M. B. et al., Biotechnol. Progr., 1999, 15: 1046–1052). In sum, due to inherent disadvantages of introducing several genomes into the host cells, a multi-protein expression system where all the proteins are obtained from the same genome is much more efficient.
With variable success, supplementing production of a protein of interest with molecular chaperones was widely used for improving protein solubility in the E. coli expression system. In this system a protein of interest was provided on a separate plasmid genome from molecular chaperones, which were provided on one or more plasmids.
Obtaining a recombinant protein of interest at a high expression level in a soluble form is the best possible scenario, however this is not always achievable. Sometimes substantial part of the protein is soluble, but the expression level is low. Very often the expression level is high, but the protein is insoluble. Many heterologous proteins are degraded by proteases and fail to achieve high expression levels. Thus, advantages of inclusion body formation is that it protects the expressed protein against degradation by proteases in host cells, can achieve high expression levels and allows separation of the inclusion body by centrifugation from the other proteins. In order to obtain the desired biologically active protein, however, it is necessary for the inclusion body to be denatured and solubilized, followed by renaturation (refolding). This solubilization-renaturation process is performed by repeated trial and error for individual proteins, but often fails to achieve satisfactory recovery rates. In some cases, renaturation is not always possible. Well-established means for solving such problems of insolubilization and degradation of expression products have not been found. Protease mutants of E. coli and baculovirus have been produced in order to reduce the degradation of foreign proteins (Suzuki T. et al., J. Gen. Virol., 1997, 78:3073–3080). However, attempts to mass-produce biologically active proteins have not always been altogether successful. In order to solve this problem, co-expression of chaperones and the like has been known. It may result in production of a soluble correctly folded protein, which is less prone to protein degradation, or in inclusion bodies, which may contain largely correctly folded protein.
Most of the studies on protein solubility improvement using molecular chaperones have been performed in E. coli. In E. coli, DnaK, DnaJ and GrpE chaperones cooperatively act in protein folding. These chaperones belong to Hsp70 molecular chaperone system, which is characteristic for nearly all types of prokaryotic (except some archaebacteria) and all types of eucaryotic cells. It has been considered that the ATP bound to DnaK is first hydrolyzed upon DnaJ binding to an unfolded protein substrate, resulting in the formation of an unfolded protein-DnaJ-DnaK (ADP binding type) complex, and thereafter ADP/ATP exchange takes place by GrpE, resulting in the release of the protein substrate from the complex (Szabo, A. et al., Proc. Natl. Acad. Sci. USA, 1994, 91: 10345–10349).
The DnaK and DnaJ genes are located at the same operon on the E. coli chromosome, while the GrpE gene is located at a site apart from the above operon. To date, there have been reported a method of coexpression of a desired protein with DnaK alone or with both DnaK and DnaJ (Blum, P. et al., BioTechnol. 1992, 10: 301–304; Perez—Perez, J. et al., Biochem. Biophys. Res. Comm. 1995, 210: 524–529); a method of coexpression of a desired protein and DnaJ alone (Japanese Patent Laid-Open No. Hei 8-308564); a method of expression of DnaK and DnaJ, and of GrpE from respectively different plasmids (Caspers, P. et al., Cell. Mol. Biol. 1994, 40: 635–644); and a method of independent expression of DnaK and DnaJ and of GrpE from the same plasmid using the same promoter (Stieger, M. and Caspers, P., 1997, Immunology Methods Manual: 39–44). However, these methods have the drawbacks described below.
Specifically, DnaK, DnaJ and GrpE, which act in cooperation with each other, are expected to be more effective when coexpressed, and it is very likely that their inherent chaperone function is not fully exhibited simply when DnaK alone or only DnaK and DnaJ are expressed. Also, in a method in which DnaK and DnaJ, and GrpE, are expressed from the respectively different plasmids, since it is difficult for a total of three plasmids, including the expression plasmid for the desired protein, to coexist in E. coli, the gene for GrpE and the gene for the desired protein are placed on a single plasmid, which in turn necessitates that the expression plasmids be constructed to adapt to individual desired proteins. Moreover, since the same promoter is used for expression of GrpE and the desired protein, the expression of the desired proteins cannot be increased to sufficient levels. Further, in the method in which DnaK, DnaJ, and GrpE are independently expressed from the same plasmid using the same promoter, another problem that arises is plasmid stability because of the presence of two units of the same promoter.
There have been reported a number of successful cases of solubilization of foreign proteins that otherwise remain insolubilized in E. coli by coexpression of the foreign protein and GroEL and GroES. Examples thereof include, for instance, tyrosine kinase (Caspers, P. et al., Cell Mol. Biol. 1994, 40: 635–644; Amrein, K. E. et al., Proc. Natl. Acad. Sci. USA 1995, 92: 1048–1052); glutamate racemase (Ashiuchi, M. et al., J. Blochem. 1995, 117: 495–498); and dihydrofolate reductase (Dale, G. E. et al., Protein Eng. 1994, 7: 925–931). Other reported cases include improvement of solubility of human growth hormone by coexpression of DnaK (Blum, P. et al., Biotechnol. 1992, 10: 301–304), transglutaminase solubilization by coexpression of DnaJ (Japanese Patent Laid-Open No. Hei 8-308564), and tyro sine kinase solubilization by coexpression of DnaK, DnaJ and GrpE (Caspers, P. et al., 1994, Cell Mol. Biol. 40, 635–644).
A majority of studies on constructing expression systems supplementing protein expression with molecular chaperones pertains to E. coli vectors. However, improved performance of eucaryotic expression systems by supplementing them with molecular chaperones has been reported.
Like in bacteria, in eucaryotic cells, the role of molecular chaperones in protein folding is well documented (Naylor D. J. and Hartl F. U., Biochem. Soc. Symp., 68: 45–68, 2001). Ubiquitous Hsp70 chaperone systems play a pivotal role in folding a large variety of proteins in multiple compartments of eucaryotic cells. The Hsp70 system is composed of Hsp70 (DnaK-like) and Hsp40 (DnaJ-like) chaperones. Like in E. coli, interaction with DnaJ homologues is essential for full activity of Hsp70. Characterized by the presence of a highly conserved 70-amino acid J domain, Dna-J homologues activate the ATPase activity of Hsp70 proteins and stabilize their interaction with unfolded substrates. There are heat-induced (Hsp70) and constitutively expressed (Hsc70) chaperones. Numerous studies have shown that DnaJ-like proteins can target substrates to Hsp70 or recruit Hsp70 proteins to substrates. In each eucaryotic cell there are a large number of Hsp70 and Hsp40 homologues, at least some of which are believed to have one or more preferred partners. For example, in the cytoplasm Hsc70 preferably interacts with constitutively expressed Hsp40 homologue dj2 (Shen et al., J. Biol. Chem., 277: 15947–15956, 2002), however, Hsp70 endoplasmic reticulum homolog Bip is likely to interact with Hsp40 homologs resident in the endoplasmic reticulum, for example HEDJ (ERdj3), ERdj4 and ERdj5 (Shen Y. et al., J. Biol. Chem., 277: 15947–15956, 2002; Cunnea P. M et al., J. Biol. Chem., 278: 1059–1066, 2003). In addition to the ubiquitous Hsp70 system, there are many other chaperones interacting with certain subsets of proteins.
For example, PDI and ERp57 participate in folding of proteins containing disulfide bonds, PPI in folding proteins containing prolin residues, Hsp90 is essential for folding steroid receptors. Lectin chaperones calreticulin and calnexin act in concert with UDP-glycoprotein glycosyl transferase, mannosidase I, glucosidase II and ERp57 to achieve quality control of glycoprotein folding in the endoplasmic reticulum (Schrag J. D. et al., Trends in Biochemical Sciences, 28: 49–57, 2003).
Hsp70 chaperone system can facilitate folding of a large variety of proteins, however for some proteins additional chaperones are required to extend protein folding capacity of the basic Hsp70 system. Hsp90 chaperone can be recruited to Hsp70 via Hop, which can bind simultaneously to both chaperones, thus facilitating their cooperative action. For example, Hsp70, Ydj1 (yeast homolog of Hsp40), Hsp90 and Hop were required for in vitro folding of functional hepadnavirus reverse transcriptase, and the fifth protein, p23, further enhanced the process. None the proteins alone, nor only two proteins in combination facilitated the protein folding except a combination of Hsp70 and Ydj1, which showed only a weak activity (Hu, S. et al. J. Virol., 76: 269–279, 2002). However, it has to be noted that a combination of human Hsp70 and yeast Ydj1 is unlikely to be optimal, as they come from distant organisms. It is known that Hsp70-like chaperones have preferred Hsp40-like chaperones partners even when they are derived from the same organisms, therefore the activity of the basic Hsp70 chaperone system could be underestimated in this experiment.
In addition to Hsp70 chaperone system, many other chaperones can contribute to protein folding, for example proteins regulating activity of Hsp70 chaperone system, comprising proteins belonging to groups of Hip and BAG-1; chaperonins and co-chaperonins comprising Hsp60, Hsp10, CCT, prefoldin (GimC); small Hsps, comprising Hsp24, Hsp25, Hsp27, Hsp28; PPIases (immunophilins); eucaryotic trigger factor homologues of nascent polypeptide-associated complex such as alpha and beta NAC. Of particular interest to obtaining soluble overexpressed proteins is the Hsp100 group of molecular chaperones, for example yeast Hsp104 or plant Hsp101 which are similar to E. coli CLPs. Hsp100s do not prevent protein aggregation of misfolded protein, however in concert with Hsp40 and Hsp70, Hsp104 can reactivate proteins that have been allowed to aggregate. Hsp104 can not cooperate with E. coli DnaK system, however it is compatible with mammalian Hsp70 system (Glover, J. R. and Lindquist, S., Cell, 94: 73–82, 1998).
There are several reports on improving protein folding and activity using molecular chaperones in higher eucaryotic expression systems. However, no convenient vector system, allowing delivery of a chaperone in the same genome with a protein of interest has been developed.
In yeast, overexpression of Bip and/or PDI has been demonstrated to facilitate secretion of single-chain antibody fragments (Shusta, E. V., et al., Nature Biotechnology, 16: 773–777, 1998). Bip and PDI acted synergistically, though a noticeable effect was also achieved with Bip or PDI alone. First, a gene encoding a protein of interest was stably inserted into the yeast genome, and polynucleotide sequences encoding Bip and PDI were provided on 2 plasmids, which were delivered into cells by co-transfection. Several times improvement was achieved in the production of secreted protein product. However the system is complex. It required the steps of incorporation of a protein of interest into the yeast genome, co-transfection of the plasmids with chaperone, and screening yeast recombinants expressing them at a desirable level. It is unclear if the obtained yeast strain is suitable for a scaled up protein production, as the plasmids were maintained in the episomal state, which is known to have a stability problem, well documented for 2 micron plasmid derivatives, such as was used to express PDI.
Improvement in folding of several proteins of interest was achieved by co-infecting insect cells with recombinant baculoviruses expressing the proteins of interest and recombinant baculoviruses expressing molecular chaperones. Co-expression of Bip or PDI or Hsp70 with IgG improved IgG solubility and secretion (Hsu T. A. et al., Protein Expr. Purif., 5: 595–603, 1994; Hsu T. A. et al., Protein Expr. Purif, 7: 281–288, 1996; Hsu T. A. and Batenbaugh, M. J., Biotechnol. Progr., 13: 96–104, 1997; Ailor E. and Batenbaugh, M. J., Biotechnol. Bioeng., 58: 196–203, 1998). PDI can act as a subunit of microsomal triglyceride transfer protein (MTP). Specialized human chaperone tapasin enhanced assembly of transporters associated with antigen processing-dependent and -independent peptides with HLA-A2 and HLA-B27 expressed in insect cells (Lauvau, G. et al., J. Biol. Chem., 274: 31349–31358, 1999). Co-expression of Hsp70 with Epstein-Barr virus replication protein, BZLF1 slightly improved its solubility, whereas introducing additional chaperones Hsp40 or its homologue Hsdj increased BZLF1 solubility 8 times (Yokoyama N. et al., Biochim. Biophys. Acta, 1493: 119–124, 2000). Co-expression of lectin chaperone calnexin with taurin transporter improved its specific activity by 53% (Miyasaka, T. et al., Protein Expr. Purif., 23: 389–397, 2001). In a separate study, co-expression with calnexin was used to improve expression of correctly assembled Shaker potassium channel in insect cells (Higgins M. K. et al., Biochim. Biophys. Acta, 1610: 124–32, 2003) Co-expression with calnexin, calreticulin, Bip and foldase (ERp57) was employed to improve expression of functional cocaine-sensitive serotonine transporter (Tate C. G. et al., J. Biol. Chem., 274: 17551–17558, 1999). In this study co-expression with calreticulin or Bip or calnexin had a positive effect, which was most pronounced with calnexin, resulting in nearly 3 times improvement in the specific activity. In another study, co-expression of calreticulin with lipoprotein lipase resulted in 9-fold increase in its enzymatic activity, however co-expression with calreticulin was less effective (Zhang L. et al., J. Biol. Chem., epub ahead of print, May 9, 2003). It appears that the degree of positive effect in co-expression experiments with particular molecular chaperones largely depends on the nature of a protein of interest. In most of these papers a positive effect on the folding of proteins of interest was noted, however typically a majority of overexpressed protein of interest remained insoluble. This could be due to a) often providing only one, or at best two molecular chaperones, and b) inherent disadvantages of co-infection method employed in these studies.
Studies on employing molecular chaperones for improved folding of proteins of interest in mammalian cells are few and suffer from the same drawbacks. No improvement in recombinant protein secretion was observed when Bip or PDI were overexpressed in mammalian cells (Dorner A. J. and Kaufman, R. J., Biologicals, 22: 103–112, 1994; Davis, R., Biotechnol. Progr., 16: 736–743, 2000). This differs from the discussed above data on the positive effects of overexpression of PDI in yeast cells or in insect cells co-infected with recombinant baculoviruses. However, different recombinant proteins and host cells were used in these studies, so the data cannot be directly compared. It is possible that level of PDI or Bip activity may not necessarily be a factor limiting recombinant protein folding in the mammalian cells used in these studies. Interestingly, overexpression of PDI can increase longevity of both mammalian cells (Kitchin, K and Flickinger, M. C., Biotechnol. Progr., 11: 565–574, 1995) and insect cells infected with recombinant baculoviruses.
Human Hsp40, or Yeast Hsp 104 or E. coli GroEL reduced protein aggregate formation and cell death caused by accumulation of intracellular inclusions in COS-7 cells (Bao Y. P. et al., J. Biol. Chem., 277: 12263–12269, 2002). The aggregates were intranuclear poly-A binding protein with an expanded polyalanine stretch, which is used as a model system of oculopharyngeal muscular dystrophy. These chaperones also reduced aggregation of green fluorescent protein provided with long polyalanine stretches.
To summarize, overexpression of molecular chaperones can improve protein folding and significantly increase yield of biologically active protein of interest. This was demonstrated in numerous studies performed in bacterial, yeast, insect and mammalian cells. Chances that the majority of an overexpressed cytoplasmic protein of interest could be produced in a biologically inactive form of inclusion bodies could be as high as about 50% and are likely to be more than 50% if a protein requires processing in the endoplasmic reticulum. Apparently, this justifies routine use the protein expression systems supplemented with molecular chaperones. Why would anybody use a system without molecular chaperones, if the system with molecular chaperones were working just as well in every respect, and in addition were likely to increase yield of a biologically active proteins? The explanation is that systems supplemented with chaperones are cumbersome and unreliable, and therefore they are very rarely used. For the same reason few such systems are commercially available. Therefore, typically, fast and convenient vectors without the chaperones are used, with the hope that the protein of interest will be soluble. If it is not, a time consuming trial and error approach for denaturing and refolding insoluble protein is applied. If that does not work, many researchers are likely to modify the protein sequence and start the protein expression work from the scratch or drop the protein, rather than apply molecular chaperones, as the chaperones systems are either not available or cumbersome or require additional expertise.
Insect cells are of growing importance for the production of recombinant proteins. A convenient and versatile baculovirus vector system using insect cells has been developed. Although information on the physiology of insect cells is rather scarce, vaccines produced via baculovirus recombinant techniques are generally well accepted. Recombinant immunodeficiency virus type I, parvovirus B19 and H5 influenza vaccines based on baculovirus-expressed proteins have been tested in clinical trials (Treanor J. J. et al., Vaccine, 19: 1732–1737, 2001).
Scale-up suspension cultures offer the best possibility for mass protein production. In large-scale production (see Tramper et al., Rec. Adv. Biotech., 1992, 263–284; Power and Nielsen, Cytotechnology 20: 209–219, 1996), special emphasis should be given to factors influencing cell growth and virus production. Variations in such factors greatly influence the final level of recombinant protein production.
Baculoviruses are characterized by rod-shaped virus particles which are generally occluded in occlusion bodies (also called polyhedra). The family Baculoviridae can be divided in two subfamilies: the Eubaculovirinae comprising two genera of occluded viruses—nuclear polyhedrosis virus (NPV) and granulosis virus (GV)—and the subfamily Nudobaculovirinae comprising the nonoccluded viruses. The cell and molecular biology of Autographa californica (Ac)NPV has been studied more in detail.
Many proteins have been expressed in insect cells infected with a recombinant baculovirus encoding that protein. Encoding means that such viruses are provided with a nucleic acid sequence encoding a heterologous protein and often are further provided with regulating nucleic acid sequences, such as a promoter. Most often, the polyhedrin promoter is used to express a foreign gene but the p10 promoter is equally well suited and used as well.
Several cell-lines are available for infection with recombinant baculovirus. The cell-line SF-21 was derived from ovarial tissue of the fall armyworm (Spodoptera frugiperda). A clonal isolate, SF-9, available from the American Type Culture Collection (CRL 1711), is more or less a standard cell-line for in vitro production of recombinant virus and is said to be superior in producing recombinant virus. Other cell-lines are, for example, the Hi-Five cell-line and the Tn-368 and Tn-368A cell-lines obtained from the cabbage looper (Trichoplusia ni). The most widely used media in which insect cells grow include TC-100, TNM-FH, BML-TC/10, and IPL-41. These media are usually supplemented with more or less defined components, such as mammalian sera, in particular, fetal calf serum. Serum replacements have also been applied to insect-cell culture, and serum-free media, such as Ex-cell 400 and Sf900 are commercially available.
Insect cells, in general, grow on solid supports as well as in suspension, but are reported to give higher yields of virus when grown on solid supports. Infection is most efficient when cells are infected in the exponential growth phase. The amount of polyhedra and virus produced per cell, however, does not vary significantly between cells infected during different stages in the cell cycle. Cell density has a great influence on virus production. Insect cells can show a form of contact inhibition resulting in reduced virus production at higher cell densities.
The initial multiplicity of infection (“m.o.i.” or “MOI”), which is the number of infectious viruses per cell, generally influences both the fraction of infected cells and the number of polyhedra per cell at the end of infection. a study (Licari and Bailey, Biotech. Bioeng., 37:238–246, 1991) of a recombinant baculovirus expressing beta-galactosidase, Sf-9 cells were infected with m.o.i. values between 0 and 100. The beta-galactosidase yield increased and cell density decreased with increasing m.o.i. It is generally thought that increasing or decreasing m.o.i. has only a limited affect on the maximum achievable yield of a recombinant protein per infected cell. Choosing low m.o.i., however, allows reduction of virus stock needed for infection and minimizes the risk of the generation of defective interfering particles of baculovirus. If a batch culture of insect cells is infected at high m.o.i. (>5), the ensuing infection process will be essentially synchronous, i.e., all cells will go through the infection cycle simultaneously.
It is necessary to know the titer of the virus stocks in order to be able to plan experiments with desired m.o.i. Methods for determining titer of the virus stocks are well developed and kits designed to simplify this essential procedure are marketed by several companies.
Through designing mathematical models, it is thought possible to predict complex behaviors such as those observed when infecting cells at low m.o.i. or when propagating virus in a continuous culture system. A purely empirical analysis of the same phenomena is considered very difficult, if not impossible. At present, three models are known: the Licari & Bailey, the de Gooijer and the Power & Nielsen model. These are, despite their complexity and the effort that has gone into developing them, all first generation models, postulating about the behavior of baculoviruses expressing a model recombinant protein (beta-galactosidase) expressed under control of the polyhedrin promoter. They summarize, to a large extent, our present quantitative understanding of the interaction between baculovirus and insect cells, when looked upon as a black box system, with disregard to DNA and RNA accumulation and the infection cycle. The binding and initial infection processes are still quantitatively poorly understood and further work in this area is much needed. An expression system involving co-infections with several types of recombinant baculoviruses is even more difficult to describe as it provides another level of complexity.
The present invention provides compositions and methods to improve yield of naturally folded protein of interest by coordinated expression of two or more molecular chaperones with a protein of interest. The invention provides other advantages as well.